The invention concerns regulatory elements of the expression of IL-16, genomic nucleic acids, cDNA and mRNA that code for polypeptides with IL-16 activity, processes for the production thereof and their use.
IL-16 (interleukin-16) is a lymphokine that is also denoted xe2x80x9clymphocyte chemoattracting factorxe2x80x9d (LCF) or xe2x80x9cimmunodeficiency virus suppressing lymphokinexe2x80x9d (ISL). IL-16 and its properties are described in WO 94/28134, the International Application PCT/EP96/01486 as well as by Cruikshank, W. W., et al., Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 5109-5113 and Baier, M., et al., Nature 378 (1995) 563. The recombinant production of IL-16 is also described therein. According to this IL-16 is a protein with a molecular mass of 13,385 D. Cruikshank also found that ISL elutes as a multimeric form with a molecular weight of 50-60 or 55-60 kD in molecular sieve chromatography. The chemoattractant activity is attributed to this multimeric form which is a cationic homotetramer (product information AMS Biotechnology Ltd., Europe, Cat. No. 11177186). Baier describes a homodimeric form of IL-16 with a molecular weight of 28 kD. However, the chemoattractant activity described by Cruikshank et al., in J. Immunol. 146 (1991) 2928-2934 and the activity of recombinant human IL-16 described by Baier are very low.
The object of the present invention is to provide regulatory elements of IL-16 expression, to improve the activity of IL-16 and to provide forms of IL-16 which exhibit low immunogenicity and are advantageously suitable for therapeutic use.
The object of the invention is achieved by a nucleic acid with which expression of a polypeptide having interleukin-16 activity can be achieved or regulated in a eukaryotic host cell, wherein the said nucleic acid
a) corresponds to the DNA sequence SEQ ID NO:1 or its complementary strand;
b) hybridizes under stringent conditions with the DNA of sequence SEQ ID NO:1, preferably with nucleotides 1-6297 of SEQ ID NO:1;
c) or is a nucleic acid sequence which, if there was no degeneracy of the genetic code, would hybridize under stringent conditions with the nucleic acid sequences defined by a) or b),
d) and, if it codes for a polypeptide having IL-16 activity, has a length of at least 1179 coding nucleotides.
A preferred sequence is the cDNA sequence shown in SEQ ID NO:6, the complementary strand thereof or a sequence which under stringent conditions hybridizes with the sequence SEQ ID NO:6. SEQ ID NO:5 and the plasmid pCI/IL16 PROM also describe the genomic DNA of IL-16 and contain the introns and exons each parity or completely.
Such a nucleic acid preferably codes a polypeptide with IL-16 activity, particularly preferably the natural IL-16 of primates such as human IL-16 or IL-16 of a species of monkey or another mammal such as e.g. mouse.
It surprisingly turned out the FIG. 2 of WO 94/28134 does not describe the complete sequence of IL-16. The start codon xe2x80x9cATGxe2x80x9d of the precursor form of the protein does not begin with nucleotide 783. The sequence has yet more differences to FIG. 2 of WO 94/28134. These are for example nucleotide substitutions (313 G by A, 717 C by A, 1070 G by T).
The sequence of IL-16 can differ to a certain extent from protein sequences coded by such DNA sequences. Such sequence variations can be amino acid substitutions, deletions or additions. However, the amino acid sequence of IL-16 is preferably at least 75% especially preferably at least 90% identical to the amino acid sequence of IL-16. Variants of parts of the amino acid sequence or nucleic acid sequence are for example described in the International Patent Application Nos. PCT/EP96/01486, PCT/EP96/05662 and PCT/EP96/05661.
Nucleic acids within the sense of the invention are for example understood as DNA, RNA and nucleic acid derivatives and analogues. Preferred nucleic acid analogues are those compounds in which the sugar phosphate backbone is replaced by other units such as e.g. amino acids. Such compounds are denoted PNA and are described in WO 92/20702. Since for example PNA-DNA bonds are stronger the DNA-DNA bonds the stringent conditions for PNA-DNA hybridization described in the following are not applicable. Suitable hybridization conditions are, however, described in WO 92/20703.
SEQ ID NO:6 describes the cDNA derived from the mRNA. The cDNA is suitable, for instance, for the determination of the corresponding RNA in tissue fluids and body fluids of mammals and humans. The cDNA is preferably used, however, for the expression of full length IL-16 in prokaryotes, preferably in E.coli. For that purpose the cDNA is inserted into an appropriate vector, transformed into a prokaryotic host cell, said host cell is cultivated, and, after cultivation, IL-16 is isolated. This can be done according to the methods known to one skilled in the art. If the protein is not secreted but obtained within the cell as a denatured insoluble protein (inclusion bodies), solubilisation and naturation must be carried out thereafter. These methods are also known to one skilled in the art.
SEQ ID NO:7 describes the amino acid sequence of IL-16 in its precursor form, which is also a subject matter of the invention.
The term xe2x80x9cIL-16xe2x80x9d within the sense of the invention is understood as a polypeptide with the activity of IL-16. IL-16 preferably exhibits the effect stated in the International Patent Application No. PCT/EP96/01486 or it stimulates cell division according to WO 94/28134.
IL-16 binds to CD4+ lymphocytes and can suppress the replication of viruses such as for example HIV-1, HIV-2 and SIV. The function of IL-16 is not limited by its presentation in the MHC complex.
IL-16 in particular exhibits one or several of the following properties:
binding to T cells via the CD4 receptor,
stimulating the expression of the IL-2 receptor and/or HLA-DR antigen on CD4+ lymphocytes,
stimulating the proliferation of T helper cells in the presence of IL-2,
suppressing the proliferation of T helper cells stimulated with anti-CD3 antibodies,
suppressing the replication of viruses preferably HIV-1, HIV-2 or SIV.
The term xe2x80x9chybridizing under stringent conditionsxe2x80x9d means that two nucleic acid fragments hybridize with one another under standardized hybridization conditions as for example described in Sambrook et al., xe2x80x9cExpression of cloned genes in E. colixe2x80x9d in Molecular Cloning: A laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, U.S.A. Such conditions are for example hybridization in 6.0xc3x97SSC at about 45xc2x0 C. followed by a washing step at 2xc3x97SSC at 50xc2x0 C. To select the stringency, the salt concentration in the washing step can be selected for example between 2.0xc3x97SSC at 50xc2x0 C. for low stringency and 0.2xc3x97SSC at 50xc2x0 C. for high stringency. In addition the temperature in the washing step can be varied between room temperature ca. 22xc2x0 C. for low stringency and 65xc2x0 C. for high stringency.
A xe2x80x9cregulatory elementxe2x80x9d is understood as a DNA sequence which regulates the expression of genes (e.g. promoter, attenuator, enhancer). A promoter is understood as a cis-acting DNA sequence which is usually 80-120 base pairs long and is located 5xe2x80x2 upstream of the initiation site of the gene to be expressed. A promoter is in addition characterized in that RNA polymerase can bind to it and can initiate the correct transcription. A preferred DNA fragment with promoter activity spans nucleotides 2053-3195 of SEQ ID NO:1.
An enhancer is usually understood as a cis-acting DNA sequence of ca. 50-100 bp in length which is of paramount importance for an efficient transcription. Enhancer sequences work independently of orientation and position.
An intron is understood as a nucleotide sequence which is present in eukaryotic genes and is transcribed into pre-mRNA and is removed from the mRNA in a further step (splicing). The IL-16 gene contains several introns and exons which are described in SEQ ID NO:1, pCI/IL16 PROM and/or SEQ ID NO:5.
Plasmid pCI/IL-16 PROM contains a sequence upstream of SEQ ID NO:5. SEQ ID NO:5 describes the 3xe2x80x2 terminal part of the genomic DNA whereas the plasmid describes the 5xe2x80x2 terminal part. Both sequences overlap in the region of nucleotide 1 to nucleotide 87 of SEQ ID NO:5. Thus the plasmid contains the IL-16 sequence 5xe2x80x2 upstream of nucleotide 87 of SEQ ID NO:5. These are coding sequences and regulatory elements as well as one or several introns. In the first intron of SEQ ID NO:5 about 600 base pairs are missing at the position denoted xe2x80x9cNxe2x80x9d. These nucleotides can either be deleted or filled up by any nucleotides. However, it is important that the intron/exon junctions remain correct. The order of these base pairs is shown in SEQ ID NO:1.
A further subject matter of the invention are regulatory elements of the expression of IL-16 (in particular promoter and enhancer elements as they are present on the plasmid pCI/IL-16 PROM or in SEQ ID NO:1/SEQ ID NO:5 or can be derived therefrom). Promoter elements are at the 5xe2x80x2 end upstream of exon 1. The enhancer elements are on the 5xe2x80x2 side of the IL-16 gene to be expressed in the said plasmid as well as at the 3xe2x80x2 end of SEQ ID NO:1/SEQ ID NO:5.
The regulatory elements according to the invention are particularly suitable for the expression of polypeptides with IL-16 activity in eukaryotic cells. The regulatory elements are, however, also suitable for expression of other genes in eukaryotic cells. The regulatory elements are particularly advantageous for expression in lymphocytes in particular in T lymphocytes and cells or cell lines derived therefrom. Suitable regulator sequences can be selected as described in example 7.
IL-16 is preferably recombinantly produced in eukaryotic host cells. Such production methods are known to a person skilled in the art and are for example described in EP-B 0 148 605. However, in order to obtain the forms of IL-16 according to the invention by recombinant production in a defined and reproducible manner, additional measures have to be taken beyond the processes for recombinant production familiar to a person skilled in the art. For this a DNA is firstly prepared which is able to produce a protein that has the activity of IL-16. The DNA is cloned into a vector that can be transferred into a host cell and can be replicated there. Such a vector contains regulatory elements that are necessary to express the DNA sequence in addition to the IL-16 sequence. One or several regulatory elements contained in SEQ ID NO:1 are preferably used. Such a nucleic acid which contains the regulatory elements is transferred into a vector which is capable of expressing the DNA of IL-16. The host cell is cultured under conditions that are suitable for vector amplification and IL-16 is isolated. In this way suitable measures ensure that the protein can adopt an active tertiary structure in which it exhibits IL-16 properties.
A lymphoid expression cell line is preferably used instead of the usual host cells (COS, CHO). In this connection IL-16 may be processed into the active shortened form.
In this process it is not necessary that the expressed protein contains the exact IL-16 amino acid sequence from SEQ ID NO:7. Proteins are equally suitable which contain essentially the same sequence and have analogous properties. A eukaryotic expression using the regulatory elements and/or the genomic DNA of IL-16 ensures that IL-16 is correctly processed. In this way a protein is obtained in a recombinant manner which is essentially or completely identical to natural IL-16.
The nucleic acid sequence of the protein can be modified. Such modifications are for example:
Modification of the nucleic acid in order to introduce various recognition sequences of restriction enzymes to facilitate the steps of ligation, cloning and mutagenesis
modification of the nucleic acid to incorporate preferred codons for the host cell
extension of the nucleic acid by additional operator elements in order to optimize the expression in the host cell.
In addition the expression vectors usually contain a selectable marker in order to select the transformed cells. Such selectable markers are for example the DHFR gene, the resistance genes for ampicillin, chloroamphenicol, erythromycin, kanamycin, neomycin and tetracyclin (Davies et al., Ann. Rev. Microbiol. 32 (1978) 469). Selectable markers which are also suitable are the genes for substances that are essential for the biosynthesis of substances necessary for the cell such as e.g. histidine, tryptophan and leucine.
Further genetic engineering methods for the construction and expression of suitable vectors are described in J. Sambrook et al., Molecular Cloning: A laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, N.Y.
Recombinant IL-16 can be expressed in eukaryotic cells such as for example CHO cells, yeast or insect cells. CHO cells, COS cells or host cells derived from lymphocytes (preferably from T lymphocytes) are preferred as the eukaryotic expression system. Expression in yeast can be achieved by means of three types of yeast vectors: integrating YIP (yeast integrating plasmids) vectors, replicating YRP (yeast replicon plasmids) vectors and episomal YEP (yeast episomal plasmids) vectors. More details of this are for example described in S. M. Kingsman et al., Tibtech 5 (1987) 53-57).
A further subject matter of the invention is a eukaryotic host cell which is transformed or transfected with a nucleic acid that codes for an IL-16 polypeptide according to the invention in such a way that the host cell expresses the said polypeptide. Such a host cell usually contains a biological functional nucleic acid vector, preferably a DNA vector e.g. a plasmid DNA that contains this nucleic acid.
A further subject matter of the invention is human interleukin-16 or interleukin-16 from primates preferably human IL-16 which can be obtained essentially free of other human proteins as a correctly processed product of a eukaryotic expression. IL-16 is a protein that occurs as a monomer or as a multimer composed of monomeric IL-16 (denoted subunits in the following). The molecular weight of a monomeric IL-16 subunit is preferably ca. 14 kD. In addition a monomeric IL-16 polypeptide is preferred which cannot be cleaved into further subunits.
It surprisingly turned out that the nucleic acid and protein sequence of IL-16 described in WO 94/28134 do not correspond to the natural human sequences. This is merely an IL-16 fragment. However, for therapeutic use it is preferable to use a correctly processed protein which is either identical to the natural protein or only differs slightly from the natural protein and exhibits at least a comparable activity and hence low immunogenicity.
Within the sense of the invention the nucleic acid sequence of IL-16 can contain deletions, mutations and additions. An IL-16 (monomeric form, subunit) that is coded by such a nucleic acid can be multimerized in a preferred embodiment. In this way the activity of IL-16 can be increased. Such multimeric forms are preferably dimeric, tetrameric or octameric forms.
In a further preferred embodiment polypeptides of the invention can additionally contain a defined content of metal ions wherein the number of metal ions per subunit is preferably 0.5 to 2.
Within the sense of the invention many metal ions are suitable as the metal ions. Alkaline earth metals as well as elements of side groups have proven to be suitable. Particularly suitable are alkaline earth metals, cobalt, zinc, selenium, manganese, nickel, copper, iron, magnesium, potassium, molybdenum and silver. The ions can be monovalent, divalent, trivalent or quadrivalent. Particularly preferred are divalent ions. The ions are preferably added as solutions of MgCl2, CaCl2, MnCl2, BaCl2, LiCl2, Sr(NO3)2, Na2MoO4, AgCl2.
Such multimeric forms and forms of IL-16 containing metal ions are described in the International Patent Application No. PCT/EP96/05661.
The polypeptide according to the invention can be produced in such a way that a eukaryotic host cell which is transformed or transfected with a nucleic acid according to the invention is cultured under suitable nutrient conditions and the desired polypeptide is optionally isolated. If the polypeptide is to be produced in vivo as part of a gene therapy treatment, the polypeptide is of course not isolated from the cell.
In addition the invention concerns a pharmaceutical composition which contains a polypeptide according to the invention in an adequate amount and/or specific activity for a therapeutic application as well as optionally a pharmaceutical suitable diluent, adjuvant and/or carrier.
The polypeptides according to the invention are particularly suitable for the treatment of pathological states that have been caused by viral replication especially retroviral replication and for immunomodulation. Such therapeutic applications are also described in WO 94/28134 as well as in the International Patent Application No. PCT/EP96/01486. Diagnostic test procedures are also described in the latter.
The polypeptides according to the invention can preferably be used for immunosuppression. This immunosuppression is preferably achieved by inhibiting the helper function of THO and/or TH1 and TH2 cells. The polypeptides according to the invention are therefore of therapeutic value in all diseases in which an immunodys-regulatory component is postulated in the pathogenesis, in particular a hyperimmunity. Diseases which can be treated with IL-16 can be diseases in cardiology/angiology such as myocarditis, endocarditis and pericarditis, in pulmonology these are for example bronchitis, asthma, in hematology autoimmune neuropenia and graft rejection, in gastroenterology chronic gastritis, in endocrinology diabetes mellitus type I, in nephrology glomerulonephritis, diseases in the field of rheumatoid diseases, diseases in opthalmology, in neurology such as multiple sclerosis, in dermatology such as eczema. The polypeptides according to the invention can in particular be used in autoimmune disease, allergies and to avoid graft rejections.
A further subject matter of the invention is the use of nucleic acids according to the invention in the field of gene therapy. Vector systems that are suitable for this are for example retroviral or non-viral vector systems.
The following examples and publications as well as the sequence protocol are intended to elucidate the invention, the scope of which is characterized by the patent claims. The methods described are to be understood as examples which also after modifications still describe the subject matter of the invention.
The plasmid pCI/IL 16 PROM was deposited on the 26.03.96 under the No. DSM 10603 at the xe2x80x9cDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM)xe2x80x9d, Mascheroder Weg 1b, D-38124 Braunschweig.
Cells
Human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll Hypaque gradient centrifugation and cultivated in RPM 1640 medium supplemented with 20% fetal calf serum, 100 units/ml IL-2 and 5 xcexcg/ml phytohemagglutinin (PHA). T lymphocyte subsets were prepared as described by Ennen, J., et al., Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 7207-7211.
RNA preparations and Northern blotting
Total RNA was extracted using the RNA-Isolation Kit (Stratagene, La Jolla, U.S.). Poly (A+) RNA was isolated from total RNA with the Oligotex-dT mRNA system (Qiagen, Hilden, DE). 10 xcexcg of total RNA or 2 xcexcg of Poly (A+) RNA were loaded on a formaldehyde agarose gel and after electrophoresis blotted onto a positively charged nylon membrane (Boehringer Mannheim GmbH, DE). The IL-16 cDNA probe was generated using the PCR DIG-probe synthesis system (Boehringer Mannheim GmbH, DE) and spans the IL-16 cDNA region from nucleotide 1693 to the end of the reading frame at nucleotide 2082. Hybridizations were carried out at 58xc2x0 C. overnight followed by several high stringency washes. For detection of the signals the DIG luminescent system (Boehringer Mannheim GmbH, DE) was employed according to the manufacturer""s recommendations. The quality of RNA preparations was routinely assessed by hybridization with a 13-actin probe. The Human RNA master blot (Clontech Laboratories, Palo Alto, U.S.) was analysed with the same IL-16 cDNA hybridization probe under comparable conditions.
Reverse transcription and PCR
Identification of the 5xe2x80x2 end of IL-16 precursor mRNA was performed using the 5xe2x80x2 RACE system for rapid amplification of cDNA ends (Life Technologies, Gaithersburg, U.S.). Additional RACE experiments were carried out with parts of the CapFinder system and Marathon-Ready cDNAs from human lymph nodes, leukocytes and murine leukocytes respectively (all from Clontech Laboratories, Palo Alto, U.S.). All other cDNAs were synthesized using up to 5 xcexcg of total PBMC RNA and oligo-dT as primer (Pharmacia, Uppsala, SE).
Gel purified PCR products were ligated into the pGEM-T vector (Promega, Madison, U.S.) before determination of the nucleotide sequences according to standard methods.
Peptides and antibodies
Antibodies specific for pro-IL-16 coupled to KLH via disulfide bonds were raised in rabbits. The antisera and the peptides were obtained from the Custom peptide antibody production program (Eurogentec, Seraing, BE). Recombinant IL-16 (rIL-16His) was used to raise antisera in goats (Baier, M., et al., Nature 378 (1995) 563). Affinity purified goat anti-IL-16 antibodies were used at an IgG concentration of 0.25 xcexcg/ml in immunoblot experiments.
Immunoblots
Cell lysates were prepared by incubation of 2.5xc3x97107 cells in 400 xcexcl of solubilization buffer (20 mM Tris HCl, pH 7.5, 1% NP-40, 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethyl-sulfonyl-fluoride, 10 mM sodium fluoride, 1 mM sodium pyrophosphate, 5 xcexcg/ml aprotinin and 5 xcexcg/ml leupeptin) for 15 minutes on ice. Nuclei were removed by centrifugation and the volume was finally adjusted to 500 xcexcl with 4xc3x97SDS sample buffer. Immunoblots were carried out according to standard protocols. Antisera were used at appropriate dilutions in blocking buffer (phosphate buffered saline (PBS), pH 7.2, 5% Marvel). Finally, bound antibody was detected using the enhanced chemoluminescence (ECL) kit (Amersham, Little Chalfont, UK).
Proteolytic cleavage of pro-IL-16 in cell lysates
Purified CD8(+) cells were lysed after cultivation for 2 days by incubation in PBS-Dulbecco/1% NP-40 for 10 minutes on ice. Lysates were clarified by centrifugation and finally diluted 1:5 in PBS. The equivalent of 4.5xc3x97106 cells was incubated with 30 xcexcg rIL-16His in a volume of 66 xcexcl for 1648 hours at room temperature. Thereafter, the cleavage of rIL-16His was analysed by immunoblotting with 1:100 diluted serum 802, which recognizes the carboxyterminus of IL-16.